Recently, a novel human src-like gene, designated fyn, has been isolated and the nucleotide sequence of its coding region has been determined. Based upon nucleotide sequence information, the predicted fyn translational product is 537 amino acids in length and shares a number of structural features with p60 c-src, including identity at 337 of the 455 amino acid residuals at its carboxy terminus. In an effort to identify the fyn translational product for further study, fyn transcripts synthesized from cDNA templates were translated in vitro. The major translational product observed was a protein of 59 kd, a size in good agreement with the extent of the fyn cDNA open reading frame. Moreover, using antibodies prepared against peptides representing fyn amino and carboxy terminal coding sequences, it was possible to immunoprecipitate the 59-kd protein, designated p59 fyn, in in vitro translational products and lysates of NIH/3T3 cells transfected with constructs containing fyn cDNA in retroviral expression vectors. p59 fyn was found to possess protein-tyrosine kinase activity.